Albumin is the smallest and most abundant of plasma proteins, generally having a molecular weight of about 69,000 and constituting slightly over half of the total protein in mammalian plasma. It is synthesized in the liver and has a half-life of about four weeks. In the human body, albumin has two important roles: (a) regulating the water balance between blood and tissues; and (b) functioning as a transport molecule for various materials which are only slightly soluble in water, such as bilirubin, fatty acids, cortisol, thyroxine and any number of drugs including sulfonamides and barbiturates.
It is frequently important to determine whether patients have a deficiency of serum albumin. Patients having such a deficiency suffer from edema which is characterized by an abnormal accumulation of serous fluid. Further, albumin deficiency can limit the transport of the slightly soluble materials noted above throughout the body.
Many methods, based on a variety of principles, have been described for the measurement of serum albumin. Of these, the methods based on dye-binding techniques are especially popular because they are readily automated and provide reproducible results. Most dye-binding techniques utilize pH indicator dyes which, on binding to a protein such as albumin, undergo a color transition characteristic of the change in pH while the solution pH is maintained constant with a buffer. Representative indicator dyes which exhibit these effects are methyl orange, bromocresol purple, bromophenol blue and bromocresol green.
However, it is known in the clinical chemistry art that such indicator dyes are not exclusively specific in binding to albumin. Rather, a number of other proteins found in the body, e.g. globulins, also bind to the indicator dyes and cause a color transition. Hence, assays utilizing indicator dyes tend to be inaccurate because they are not specific enough to albumin and are highly susceptible to binding with those so-called "interfering" proteins.
Indicator dyes are useful only in narrow pH ranges. Outside those ranges, the dyes either fail to change color upon binding to protein or change color prematurely. Further, albumin assays utilizing such indicator dyes are generally carried out at relatively low pH (e.g. at about 4.0 when using bromocresol green; at about 5.2 when using bromocresol purple) in order to increase the binding of dye to albumin and to allow for a color change in the dye absorption spectrum as a result of a pKa shift. However, since the pH transitions of the more commonly used dyes, e.g. bromocresol green and bromocresol purple are at acidic pH values, non-specific binding of those dyes with protein molecules other than albumin is increased. This undesirable effect is due to the fact that most proteins are positively-charged at acidic pH values whereas the dye molecules are negatively charged. At higher pH values, most proteins are negatively charged and non-specific ionic interactions are reduced.
Hence, there is a need in the art for an assay for albumin which is highly specific for that protein and which is useful over a relatively broad pH range and particularly at alkaline pHs.